Project Summary The interplay between cellular adhesion and cellular signaling is essential for the development of all organs such as the brain, and for the functioning of systems such as the immune and nervous systems. Adhesion G- Protein Coupled Receptors (aGPCRs) are an understudied GPCR family that is thought to mediate intercellular communication. They have emerging roles in multiple cellular functions and numerous human diseases including neurological disorders and cancers. Unlike other GPCRs, aGPCRs have large extracellular regions (ECR) that are autoproteolytically cleaved from their seven-pass transmembrane regions (TM) within a conserved GPCR-Autoproteolysis INducing (GAIN) domain. The two fragments stay associated even after cleavage. It is believed that ligand binding to the ECR causes shedding of the ECR, and exposure of a short peptide that was previously hidden within the GAIN domain. This short peptide, called stachel, acts as a tethered agonist and activates the transmembrane domain. However, the molecular mechanisms underlying aGPCR activation remains poorly understood. The ultimate goal of the research proposed in this application is to understand the stepwise mechanical details of aGPCR activation that start with adhesion of the ligand to the ECR, continue with transduction of the adhesion signal from the ECR to the TM domain via the GAIN domain, and end with the activation of the TM domain by the stachel peptide. We propose three Specific Aims that are based on the major unknowns in these fundamentals steps for aGPCR activation: First, we aim to reveal if and how extracellular adhesion starts signaling. Second, we aim to understand the role of the GAIN domain in transducing the adhesion signal to the TM domain. Third, we aim to reveal the molecular details of aGPCR TM domain activation by the stachel peptide. This research has a multi-disciplinary approach where the structural and functional data performed in the PI's lab are complemented with electron microscopy, agonist/antagonist generation via protein engineering, neuronal assays and G-protein expertise provided or performed by the laboratories of close collaborators. The proposed experiments will build on exciting results obtained since we started our lab, including the establishment of a robust in vitro signaling assay, the crystal structure of a ligand- aGPCR complex, the engineering of a synthetic protein that inhibits an aGPCR function, and key advances in the purification protocols of aGPCRs. We expect that this research will provide critical insights into the mechanistic details of the aGPCR activation, helping to establish fundamental principles on intercellular communication that are vital for numerous cellular functions.